The pericentromeric heterochromatin of homologous chromosomes remains associated after centromere pairing dissolves in mouse spermatocyte meiosis

Chromosoma - In meiosis, crossovers between homologous chromosomes link them together. This enables them to attach to microtubules of the meiotic spindle as a unit, such that the homologs will be...     

Presence of macrotubules and their induction by colchicine in grasshopper spermatids

Ultrastructural studies show that groups of tubular structures of about 45 nm. in diameter appear in the cytoplasm of grasshopper spermatids. These tubules, or macrotubules, appear to have a fuzzy coat or to be twisted, and in some cases their bundles attain a length of 10 um. When grasshoppers were treated with colchicine, spermatid microtubules which composed the manchette are depolymerized and a large number of macrotubules appears. The relationship between these two types of tubules is discussed.     

Organized microtubule arrays in γ-tubulin-depleted Drosophila spermatocytes

To assess the role of γ-tubulin in spindle assembly in vivo, we have followed meiosis progression by immunofluorescence and time-lapse video microscopy in γTub23C^PI mutant spermatocytes. We have found that centrosomes associate with large numbers of astral microtubules even though γ-tubulin is severely depleted; bipolar meiotic spindles are never assembled; and later in meiosis, the microtubules get organized into a conical structure that is never observed in wild-type cells. Several lines of evidence suggest that these cones may be related to wild-type central spindles. First, they are assembled midway through meiosis and elongate during anaphase. Second, they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type spindle midzone. Third, Klp3A and Polo, two markers of the wild-type central spindle are also found around the pointed end of the mutant cones. Finally, ectopic cytokinesis furrows are often formed at the distal end of the cone. Our results suggest that microtubule polymerization or stabilization from the centrosome may be possible in a γ-tubulin-independent manner in Drosophila spermatocytes. However, γ-tubulin seems to be essential for spindle assembly in these cells. Finally, our results show that at least part of the central spindle and constriction-ring assembly machinery can operate on microtubule bundles that are not organized as bipolar spindles.     

Acetylated alpha-tubulin in microtubules during mouse fertilization and early development

alpha-Tubulin in the microtubules of mouse oocytes and embryos is acetylated in a specific spatial and temporal sequence. In the unfertilized oocyte, a monoclonal antibody to the acetylated form of alpha-tubulin is bound predominantly at the poles of the arrested metaphase meiotic spindle. The labeling intensity of the spindle microtubules is weaker as observed by immunofluorescence using oocytes double-labeled for total tubulin and acetylated alpha-tubulin, and as measured by immuno high-voltage electron microscopy (immunoHVEM) with colloidal gold; cytasters are not acetylated. At meiotic anaphase, the spindle becomes labeled, and by telophase and during second polar body formation only the meiotic midbody is acetylated. The sperm axoneme retains its acetylation after incorporation though the interphase microtubules are not detected. First mitosis follows a pattern similar to that observed at the second meiosis and during interphase only the mitotic midbodies are acetylated. After treatment with cold, colcemid, or griseofulvin, the remaining stable microtubules are acetylated, but immunoHVEM observations suggest that these fibers might not have been acetylated prior to microtubule disruption. Taxol stabilization does not alter acetylation patterns. Acetylated microtubules are not necessarily old microtubules since acetylated fibers are observed at 30 sec after cold recovery. These results show the presence of acetylated microtubules during meiosis and mitosis and demonstrate a cell-cycle-specific pattern of acetylation, with acetylated microtubules found at the centrosomes at metaphase, an increase in spindle labeling at anaphase, and the selective deacetylation of all but midbody microtubules at telophase.     

Nucleation of macrotubules on double-walled microtubule seeds

It is known that histone H1 is able to cause the formation of double-walled microtubules from microtubule protein. Now, we demonstrate that in dependence on the mass ratio H1/microtubule protein upon addition of tubulin to short pieces of double-walled microtubules either their inner or their outer wall elongates resulting in normal microtubules or in macrotubules, respectively. Because of their genesis we suggest that macrotubules like double-walled microtubules (see Unger et al., Eur. J. Cell Biol. 46, 98-104 (1988)) expose those sides of tubulin dimers at their surface which usually form the lumen face of microtubules.     

Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.     

Kinetochore orientation in mitosis and meiosis

Kinetochores are the major point of contact between spindle microtubules and chromosomes. They are assemblies of more than 50 different proteins and take part in regulating and controlling their own interaction with the spindle. We review recent advance in understanding how kinetochores are properly placed onto the chromosome, and how their interaction with the microtubules of the spindle is regulated. Kinetochore orientation in meiosis I shows some particular features, and we also discuss similarities and differences between mitosis and meiosis I.     

洋葱小孢子母细胞减数分裂过程中微管分布变化

应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察洋葱小孢子母细胞减数分裂过程中微管分布变化。减数分裂之前,小孢子母细胞中的微管较短,呈辐射状,由细胞核表面向四周扩散。减数分裂开始后,细胞质中的一部分微管蛋白聚集成纺锤体微管,控制染色体的分布。进入减数分裂I后期,纺锤体微管变为牵引染色体移向两极的着丝粒微管和连接纺锤体两极的极丝微管。之后,所有微管集中在两个核之间,构成成膜体。然后,微管解聚成微管蛋白弥散在细胞质中。减数分裂I完成后,二分体2个子细胞中的微管蛋白又聚集成2个纺锤体微管,开始减数分裂II过程。经过减数分裂II中期,2个二分体细胞中的微管再次集中在2个细胞核之间形成成膜体,隔离2个细胞核。此后,微管蛋白解聚,弥散分布在小孢子细胞质中。     

A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes

The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.     

The behaviour of kinetochore microtubules during meiosis in the fungusSaprolegnia

Summary InSaprolegnia, kinetochore microtubules persist throughout the mitotic nuclear cycle but, whilst present at leptotene, they disappear coincidently with the formation of synaptonemal complexes at pachytene and reform at metaphase I. In some other fungi chromosomal segregation is random in meiosis and non-random in mitosis. The attachment of chromosomes to persistent kinetochore microtubules in mitosis, but not meiosis, inSaprolegnia provides a plausible explanation for such behaviour. At metaphase I each bivalent is connected to the spindle by 2 laterally paired kinetochore microtubules whereas at metaphase II (as in mitosis) each univalent bears only one kinetochore microtubule, thus showing that all kinetochores are fully active at all stages of meiosis.     

Influence of vincristine on the Golgi complex of leukaemic lymphoblasts

In vitro and in vivo effects of vincristine on the Golgi complex of leukaemic lymphoblasts were studied. The cells incubated in vitro for 4 hours with vincristine of 1.25 x 10(-5) M concentration lacked microtubules, but regularly contained paracrystals and parallel arrays of macrotubules associated with ribosomes. The Golgi complex in control lymphoblasts was represented by 1-3 dictyosomes (stacks of cisternae) grouped in one area. After exposure to vincristine the dictyosomes lay at a considerable distance from each other. In many of them the cisternae were shorter than in controls and distended or transformed into large vacuoles. In cells incubated in vitro with lower concentrations of vincristine (1.25 x 10(-6) and 1.25 x 10(-7) M) and in cells obtained after the second therapeutic dose of vincristine (in the course of normal clinical treatment) neither changes in the Golgi complex nor formation of paracrystals and macrotubules were observed.     

Monastral bipolar spindles in meiosis II of male Trichosia pubescens (Sciaridae): early stages of spindle formation and chromosome orientation.

The metaphase spindle of male meiosis II in fungus gnats (Sciaridae) is one example of naturally occurring monastral bipolar spindles. To gain further insights into how the bipolar spindle is formed in the presence of only one polar center, prometaphase of male meiosis II was investigated in the sciarid Trichosia pubescens by means of anti-tubulin immunofluorescence, DAPI chromosome staining, and electron microscopy of ultrathin serial sections. The first step in spindle formation after interkinesis seems to be the organization of an astral half-spindle, probably by MTOC activity of the astral region. With the exception of the non-disjunctional X chromosome, which always lies close to the aster, the chromosomes are found to occupy various positions with respect to the astral region, revealing different orientations of their chromatid kinetochores. It was observed that some of the mal-oriented kinetochores are associated with microtubules that, due to their orientation perpendicular to the spindle axis, are unlikely to originate from the astral region. Therefore, these mal-oriented microtubules are taken as an indication of a dispersed MTOC activity near the chromosomes or at kinetochores. According to recent models of chromosome-induced spindle self-organization [e.g., Merdes et al., 1997: J. Cell Biol. 138:953-956], they could be responsible for the formation of the other (anastral) half-spindle and for amphitelic (bipolar) orientation of the chromosomes.     

The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes

Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.     

驱动蛋白Kin11调控嗜热四膜虫生殖系小核的减数分裂

驱动蛋白(kinesin)是分子马达蛋白质超家族成员,主要参与囊泡与细胞器的运输、纺锤体组装、有丝分裂和减数分裂等过程。在减数分裂期,不同驱动蛋白发挥功能的调控机制并不十分清楚。嗜热四膜虫(Tetrahymena thermophila)中含有14个驱动蛋白家族成员。其中,kinesin-6家族的唯一成员Kin11(TTHERM_00637750),在营养生长期低表达,饥饿期不表达,有性生殖期表达上调。Kin11编码1608个氨基酸,包含1个N端保守的马达蛋白结构域,C端卷曲螺旋(coiled-coil)结构域,并在N端和C端分别含有核定位信号NLS1和NLS2。Kin11在营养生长期和有性生殖期,定位在有丝分裂和减数分裂的小核和纺锤体上,并在有性生殖后期alignment阶段定位于小核上。Kin11与微管蛋白共定位于有丝分裂和减数分裂的纺锤体上。将Kin11的N端含有NLS1的1~400位氨基酸序列截短后,截断突变体定位在有性生殖减数分裂期的小核和纺锤体上。而将其C端含有NLS2的1008~1608位氨基酸残基截短后,截断突变体只能定位在有丝分裂和减数分裂后期的小核及有丝分裂的纺锤体上。敲除KIN11导致减数分裂过程中的纺锤体结构发生异常变化,小核染色体不均等分离与丢失,有性生殖发育停滞。结果表明,嗜热四膜虫驱动蛋白Kin11通过影响纺锤体结构,参与调控四膜虫生殖系小核在减数分裂过程中的正常分离。     

Nonrandom chromosome segregation in Neocurtilla (Gryllotalpa) hexadactyla: an ultrastructural study

During meiosis I in males of the mole cricket Neocurtilla (Gryllotalpa) hexadactyla, the univalent X1 chromosome and the heteromorphic X2Y chromosome pair segregate nonrandomly; the X1 and X2 chromosomes move to the same pole in anaphase. By means of ultrastructural analysis of serial sections of cells in several stages of meiosis I, metaphase of meiosis II, and mitosis, we found that the kinetochore region of two of the three nonrandomly segregating chromosomes differ from autosomal kinetochores only during meiosis I. The distinction is most pronounced at metaphase I when massive aggregates of electron-dense substance mark the kinetochores of X1 and Y chromosomes. The lateral position of the kinetochores of X1 and Y chromosomes and the association of these chromosomes with microtubules running toward both poles are also characteristic of meiosis I and further distinguish X1 and Y from the autosomes. Nonrandomly segregating chromosomes are typically positioned within the spindle so that the kinetochoric sides of the X2Y pair and the X1 chromosome are both turned toward the same interpolar spindle axis. This spatial relationship may be a result of a linkage of X1 and Y chromosomes lying in opposite half spindles via a small bundle of microtubules that runs between their unusual kinetochores. Thus, nonrandom segregation in Neocurtilla hexadactyla involves a unique modification at the kinetochores of particular chromosomes, which presumably affects the manner in which these chromosomes are integrated within the spindle.     

Abortive second meiosis detected in cytochalasin-treated eggs in androgenetic diploid Corbicula fluminea

The hermaphroditic diploid clam Corbicula fluminea reproduces by androgenesis. In the control (androgenetic development), all maternal chromosomes and maternal centrosomes at the meiotic poles were extruded as two first polar bodies and subsequently second meiosis did not occur. In eggs treated with cytochalasin D (CD) to inhibit the polar body extrusion, the second meiosis was abortive. After the first meiosis, two centrosomes at the spindle poles remained in the cytoplasm because of the effect of CD. The chromosomes divided into two groups at anaphase-I as observed in the control eggs. Two centrosomes divided into four just after the first meiosis but did not separate completely. The microtubules from the centrosomes were rather short. So at the second meiosis, two monoasters or tetrapolar spindles were formed. The fluorescence signal from microtubules of the monoaster or tetrapolar spindle was weak compared with the spindle at the first meiosis. The maternal chromosomes on the monoaster or tetrapolar spindle did not move, and became large female pronuclei. The pronuclei became the metaphase chromosomes on the spindle for the first cleavage. The present study suggests that second meiosis regulating factors may be abortive in androgenetic diploid C. fluminea.     

Bub3 Is a Spindle Assembly Checkpoint Protein Regulating Chromosome Segregation during Mouse Oocyte Meiosis

In mitosis, the spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes have been attached to the spindle microtubules and aligned correctly at the equatorial metaphase plate. The major checkpoint proteins in mitosis consist of mitotic arrest-deficient (Mad)1–3, budding uninhibited by benzimidazole (Bub)1, Bub3, and monopolar spindle 1(Mps1). During meiosis, for the formation of a haploid gamete, two consecutive rounds of chromosome segregation occur with only one round of DNA replication. To pull homologous chromosomes to opposite spindle poles during meiosis I, both sister kinetochores of a homologue must face toward the same pole which is very different from mitosis and meiosis II. As a core member of checkpoint proteins, the individual role of Bub3 in mammalian oocyte meiosis is unclear. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of Bub3 in mouse oocyte meiosis. Our data showed that overexpressed Bub3 inhibited meiotic metaphase-anaphase transition by preventing homologous chromosome and sister chromatid segregations in meiosis I and II, respectively. Misaligned chromosomes, abnormal polar body and double polar bodies were observed in Bub3 knock-down oocytes, causing aneuploidy. Furthermore, through cold treatment combined with Bub3 overexpression, we found that overexpressed Bub3 affected the attachments of microtubules and kinetochores during metaphase-anaphase transition. We propose that as a member of SAC, Bub3 is required for regulation of both meiosis I and II, and is potentially involved in kinetochore-microtubule attachment in mammalian oocytes.     

Vinblastine-induced paracrystals and unusually large microtubules (macrotubules) in rat renal cells

Summary Vinblastine sulfate was administered to adult rats by intravenous injections. Kidney cortex was fixed after 1, 2, or 5 hours of treatment and studied by routine transmission electron microscopy.In control animals, cells of distal convoluted tubules possessed numerous microtubules with an average diameter of 280 Å. In treated animals, the microtubules of these cells were reduced in number, and paracrystalline inclusions characteristic of vinblastine treatment were common. Macrotubules (570 Å average diameter) were also present and often were seen close to, or in apparent continuity with, paracrystals. Since the work of others indicates that vinblastine-induced paracrystals contain microtubular protein (tubulin), observation of continuities between paracrystals and macrotubules is interpreted as evidence that macrotubules are also composed of tubulin and that macrotubules may become incorporated into paracrystals.Unlike the ordinary microtubules of cells of the distal tubules, vinblastine-induced macrotubules exhibited cross-striations in longitudinal view and subunit structure in cross section.Macrotubules and paracrystals were also observed in cells of the proximal convoluted tubule, mesangium, glomerular endothelium, parietal epithelium of Bowman's capsule, and visceral epithelium of Bowman's capsule. Continuities between macrotubules and paracrystals, although relatively common in occurrence in distal tubule cells, were only rarely seen in the other kinds of cells examined. Acknowledgements. The authors gratefully acknowledge the technical help of Mrs. Dawn Bockus, Miss Judy Groombridge, Mrs. Jeri Hunter, Mrs. Jolan Pinter, Miss Franque Remington, Miss Mary Stewart, Miss Louise Young, Mr. Reginald Pickering, and Mr. W. J. Masten. This research was supported by N.I.H. grants AM 16 236, GM 00 100, and HE 03 174, by Institutional Cancer Grant IN-26L from the American Cancer Society, and by the Graduate School Research Fund of the University of Washington.     

Purification and characterization of oocyte cytoplasmic tubulin and meiotic spindle tubulin of the surf clam Spisula solidissima

Assembly-competent tubulin was purified from the cytoplasm of unfertilized and parthogenetically activated oocytes, and from isolated meiotic spindles of the surf clam, Spisula solidissima. At 22 degrees C or 37 degrees C, Spisula tubulin assembled into 48-51-nm macrotubules during the first cycle of polymerization and 25-nm microtubules during the third and subsequent cycles of assembly. Macrotubules were formed from sheets of 26-27 protofilaments helically arranged at a 36 degree angle relative to the long axis of the polymer and were composed of alpha and beta tubulins and several other proteins ranging in molecular weight from 30,000 to 270,000. Third cycle microtubules contained 14-15 protofilaments in cross-section and were composed of greater than 95% alpha and beta tubulins. After three cycles of polymerization at 37 degrees C, unfertilized and activated oocyte tubulin self-assembled into microtubules at a critical concentration (Ccr) of 0.09 mg/ml. At the physiological temperature of 22 degrees C, unfertilized oocyte tubulin assembled into microtubules at a Ccr of 0.36 mg/ml, activated oocyte tubulin assembled at a Ccr of 0.42 mg/ml, and isolated meiotic spindle tubulin assembled at a Ccr of 0.33 mg/ml. The isoelectric points of tubulin from both unfertilized oocytes and isolated meiotic spindles were 5.8 for alpha tubulin and 5.6 for beta tubulin. In addition, one dimensional peptide maps of oocyte and spindle alpha and beta tubulins were very similar, if not identical. These results indicate that unfertilized oocyte tubulin and tubulin isolated from the first meiotic spindle are indistinguishable on the basis of assembly properties, isoelectric focusing, and one dimensional peptide mapping. These results suggest that the transition of tubulin from the quiescent oocyte state to that competent to form spindle microtubules in vivo does not require special modification of tubulin but may involve changes in the availability of microtubule organizing centers or assembly-promoting microtubule-associated proteins.